SYNTHESIS AND BIOLOGICAL EVALUATION OF POTENT INTEGRIN LIGANDS CONTAINING A DIKETOPIPERAZINE SCAFFOLD, AND OF THEIR CONJUGATES WITH CYTOTOXIC AGENTS

A library of eight bifunctional 2,5-diketopiperazine (DKP) scaffolds, which are formally derived from 2,3-diaminopropionic acid and aspartic acid (DKP1 - DKP7) or glutamic acid (DKP8) was designed and synthesized. The scaffolds differ for the configuration at the two stereocenters and the substitution at the diketopiperazinic nitrogens. The bifunctional diketopiperazines were introduced into eight cyclic peptidomimetics containing the Arg-Gly-Asp (RGD) sequence. The resulting RGD-peptidomimetics were screened for their ability to inhibit biotinylated vitronectin binding to the purified integrins alphaVbeta3 and alphaVbeta5, which are involved in tumor angiogenesis. Nanomolar values were obtained for the RGD-peptidomimetics derived from trans DKP scaffolds (DKP2 - DKP8). Conformational studies of the cyclic RGD-peptidomimetics by 1H-NMR experiments (VT-NMR and NOESY) in aqueous solution and Monte Carlo – Stochastic Dynamics (MC-SD) simulations revealed that the highest affinity ligands display well-defined preferred conformations featuring intramolecular hydrogen-bonded turn motifs and an extended arrangement of the RGD sequence [Cbeta(Arg)-Cbeta(Asp) average distance ≥ 8.8 A]. Docking studies were performed, starting from the representative conformations obtained from the MC/SD simulations and taking as a reference model the crystal structure of the extracellular segment of integrin alphaVbeta3 complexed with the cyclic pentapeptide Cilengitide. The highest affinity ligands produced top-ranked poses conserving all the important interactions of the X-ray complex. Since alphaV integrins, which can be internalized by cells, are involved in tumor angiogenesis and are overexpressed on the surface of cancer cells, integrin ligands can be usefully employed as tumor homing peptidomimetics for site-directed delivery of cytotoxic drugs. A small library of integrin ligand - Paclitaxel conjugates 90-93 was synthesized with the aim of using the tumor-homing cyclo[DKP-RGD] peptidomimetics for site-directed delivery of the cytotoxic drug. All the Paclitaxel-RGD constructs 90-93 inhibited biotinylated vitronectin binding to the purified alphaVbeta3 integrin receptor at low nanomolar concentration and showed in vitro cytotoxic activity against a panel of human tumor cell lines similar to that of Paclitaxel. Among the cell lines, the cisplatin-resistant IGROV-1/Pt1 cells expressed high levels of integrin alphaVbeta3, making them attractive to be tested in in vivo models. Cyclo[DKP-f3-RGD]-PTX 91 displayed sufficient stability in physiological solution and in both human and murine plasma to be a good candidate for in vivo testing. In tumor-targeting experiments against the IGROV-1/Pt1 human ovarian carcinoma xenotransplanted in nude mice, compound 91 exhibited a superior activity than Paclitaxel, despite the lower (ca. half) molar dosage used.